L-Glutamate (Glu) and L-aspartate (Asp) are regarded as important excitatory amino acids (EAA) in the vertebrate nervous systems. However, their identity as the true endogenous ligands for the well characterized subtypes of EAA receptors is not established with certainty. The studies described in this proposal are designed to test the hypothesis that in addition to (or instead of) Glu and Asp, small peptides with C-terminal Glu and Asp, respectively, may be present in the brain and may act as excitatory neurotransmitters or modulators. This hypothesis is based on preliminary studies indicating that two antisera we have produced to Glu and Asp recognize,respectively. (1)Glu and Asp, (2) dipeptides with C- terminal Glu and Asp residues, (3) nonendogenous compounds which can discriminate between subclasses of EAA receptors. Thus, quisqualate (Quis) selectively blocks the immunostaining with the Glu antiserum, kainate (KA) blocks that with the Asp antiserum, while N-methyl-D-Asp (NMDA) is not recognized by either of the antisersa (a new antiserum to NMDA is in preparation). The principal goal of our studies is to use these antisera to identify and characterize putative endogenous ligands specific for the three major subtypes (Quis, KA, and NMDA) of EAA receptors in the rat brain. First, the antisera will be further characterized in terms of the molecular requirements for optimal antigen recognition, using synthetic dipeptides as well as receptor agonists and antagonists. Second, the endogenous compounds recognized by these antisera will be extracted from brain on affinity columns prepared with IgG fractions from the antisera. Finally, chromatography. Enzyme immunoassays. light and electron microscopic immunocytochemistry, and in vitro receptor activation studies will be used to monitor and complement these procedures. The results of these studies are expected to provide fundamental new insights into the molecular mechanisms of EAA neurotransmission. Identification of new endogenous ligands for EAA receptors will allow a better characterization of the neuronal pathways that use these ligands, will help in the design of new specific agonists and antagonists for EAA receptors and will provide clues for the understanding and therapy of such disease processes as brain damage following stroke and hypoglycemia, epilepsy, Huntington's chorea, and Alzheimer's disease.